This invention relates to an improved method for producing monocellular layers of cell-containing fluids, such as blood, to be analyzed by differential counting of the cells in the monolayer. More particularly, the invention relates to an improved method wherein production of a high quality monolayer is facilitated and cell distortion is minimized by spinning of a slide with the fluid sample-bearing surface thereof facing downwardly and while in a holder having integral means for shielding the sample from the buffeting effects of air currents normally generated by such spinning.
With the advent of widespread use of Automated Differential Cell counting techniques as an aid in the diagnosis of disease, various instruments have been developed for preparing the monocellular layers of biological fluids, such as blood, which are preferred for such analysis. The preparation of blood specimens by the "spinner" method to produce a generally monocellular layer of blood on a slide and prior art apparatus for practicing such methods are discussed in the following publications:
Ingram, M. and Minter, F. M.: Semiautomatic Preparation of Coverglass Blood Smears Using a Centrifugal Device, Am. J. Clin. Path., 51:214-221, 1969.
Bacus, J. W.: Erythrocyte Morphology and Centrifugal "Spinner" Blood Film Preparations, J. Histochem. Cytochem., 22: 506-516, 1974.
Wenk, R. E.: Comparison of Five Methods of Preparing Blood Smears, AJMT, 42: 71-78, 1976.
Nourbakhsh, M., Atwood, J. G., Raccio, J. and Seligson, D.: An Evaluation of Blood Smears Made by a New Method Using a Spinner and Diluted Blood, AJCP, 70: 885-892, 1978.
Various problems have been encountered in the preparation of monocellular layers of blood. One such problem is that of wide variations in the differential count depending upon where in the layer of smear the count is taken. U.S. Pat. No. 3,577,267, issued to Preston, et al. on May 4, 1971, discloses a method by which, it is stated, a uniform monolayer of flattened undamaged cells is produced. By this method, a sample-bearing slide is spun at a speed in a range of from 4,000 to 10,000 rpm for a few tenths of a second after acceleration to that range within less than ten milliseconds. Producing such acceleration, and, of course, the deceleration necessary to limit the spinning to the precise fractional second period, required a relatively high powered, heavy motor, flywheel and clutching/braking mechanism, and sophisticated time control equipment.
Another problem encountered in the preparation of monocellular layers is cell distortion resulting from the buffeting or shear effect of air currents set up by the spinning slide and holder therefor, and from resultant premature drying of the spun sample. U.S. Pat. No. 3,705,048, issued to Staunton on Dec. 5, 1972, discloses an apparatus, the objective of which is protection of the sample from undesirable buffeting and drying. In this patent the spinning slide and the holder therefor to which it is clamped are enclosed within a relatively close-fitting enclosure which reduces the amount of air which can contact the spinning sample by limiting such contact to the air within the enclosure.
Presently available apparatus for the preparation of monocellular layers is expensive, heavy, cumbersome and requires a blood sample, typically 100 microliters in volume, which must be obtained by venous puncture. The need for a blood sample of this size presents difficulties not presented where a much smaller size capillary blood sample, obtainable, for example, by a finger prick, will suffice. Obtaining a venous blood sample causes considerably more patient discomfort and requires considerably more time than does obtaining a capillary blood sample. Moreover, the requirement for a venous blood sample presents difficulties in pediatrics where infants have too little blood to spare for samples of such size.